Allelopathic potential of apple hairy root secondary metabolites - the effect of phloretin and phloridzin in vitro culture

Ograniĉenja u izuĉavanju potencijala i mehanizama alelopatskog delovanja glavnih sekundarnih metabolita korena jabuke, floretina i florizina, usled kompleksnosti interakcija u rizosferi, mogu biti prevaziĊena upotrebom kulture transformisanih korenova in vitro. Odabranim naĉinom inokulacije regenerisanih izdanaka jabuke sa Agrobacterium rhizogenes 15834, efikasno su indukovani transformisani korenovi kod sorti Melrouz, Zlatni Delińes, Ĉadel i Gloster. Smenom hranljivih podloga sa i bez auksina, po prvi put je uspostavljena permanentna in vitro kultura izolovanih transformisanih korenova jabuke. UHPLC/(+/-)HESI–MS/MS analizom je utvrĊeno da genetiĉka transformacija nije dovela do promena u sadrņaju glavnih sekundarnih metabolita korena jabuke. U eksudatu transformisanih korenova, sadrņanom u teĉnoj podlozi u kojoj su korenovi gajeni, kvantifikovani su iskljuĉivo florizin, floretin, hlorogena i kafeinska kiselina. Tretman ovim eksudatom je prouzrokovao znaĉajnu inhibiciju rastenja i razvića izdanaka i korenova klijanaca Arabidopsis thaliana. Sliĉan inhibitorni efekat ispoljili su ĉist florizin u koncentraciji od 500 μM, kao i floretin u svim testiranim koncentracijama (125-500 μM), izazivajući krupne promene u morfologiji tretiranih klijanaca, posebno korena. Velike sliĉnosti su zapaņene u obrascu promena ekspresije gena ukljuĉenih u polarni transport auksina, kao i biosintezu auksina i giberelina u korenovima klijanaca arabidopsisa tretiranih eksudatom transformisanih korenova jabuke, sa onim detektovanim kod klijanaca tretiranih 500 μM floretinom. To ukazuje na mogućnost da je floretin u eksudatu korena jabuke metabolit koji je odgovoran za alelopatske efekte, a koji bi se mogli zasnivati na promenama nivoa auksina i giberelina, kao i na interakciji ove dve grupe hormona

Allelopathic potential of apple hairy root secondary metabolites - the effect of phloretin and phloridzin in in vitro culture

Ograniĉenja u izuĉavanju potencijala i mehanizama alelopatskog delovanja glavnih sekundarnih metabolita korena jabuke, floretina i florizina, usled kompleksnosti interakcija u rizosferi, mogu biti prevaziĊena upotrebom kulture transformisanih korenova in vitro. Odabranim naĉinom inokulacije regenerisanih izdanaka jabuke sa Agrobacterium rhizogenes 15834, efikasno su indukovani transformisani korenovi kod sorti Melrouz, Zlatni Delińes, Ĉadel i Gloster. Smenom hranljivih podloga sa i bez auksina, po prvi put je uspostavljena permanentna in vitro kultura izolovanih transformisanih korenova jabuke. UHPLC/(+/-)HESI–MS/MS analizom je utvrĊeno da genetiĉka transformacija nije dovela do promena u sadrņaju glavnih sekundarnih metabolita korena jabuke. U eksudatu transformisanih korenova, sadrņanom u teĉnoj podlozi u kojoj su korenovi gajeni, kvantifikovani su iskljuĉivo florizin, floretin, hlorogena i kafeinska kiselina. Tretman ovim eksudatom je prouzrokovao znaĉajnu inhibiciju rastenja i razvića izdanaka i korenova klijanaca Arabidopsis thaliana. Sliĉan inhibitorni efekat ispoljili su ĉist florizin u koncentraciji od 500 μM, kao i floretin u svim testiranim koncentracijama (125-500 μM), izazivajući krupne promene u morfologiji tretiranih klijanaca, posebno korena. Velike sliĉnosti su zapaņene u obrascu promena ekspresije gena ukljuĉenih u polarni transport auksina, kao i biosintezu auksina i giberelina u korenovima klijanaca arabidopsisa tretiranih eksudatom transformisanih korenova jabuke, sa onim detektovanim kod klijanaca tretiranih 500 μM floretinom. To ukazuje na mogućnost da je floretin u eksudatu korena jabuke metabolit koji je odgovoran za alelopatske efekte, a koji bi se mogli zasnivati na promenama nivoa auksina i giberelina, kao i na interakciji ove dve grupe hormona.The investigation of allelopathic potential and mode of action of the main apple root secondary metabolites, phloridzin and phloretin is difficult to perform due to high complexity of rhizosphere interactions. Therefore, the generation of in vitro hairy root cultures could be an alternative system for allelopathic studies of apple. The efficient induction of hairy roots in apple cvs. Melrose, Golden Delicious, Ĉadel and Gloster was obtained using the appropriate mode of inoculation of regenerated shoots by Agrobacterium rhizogenes 15834. Permanent in vitro culture of apple autonomous hairy roots was established for the first time by cultivation on alternating growth media with and without auxins. UHPLC(+/-)HESI-MS/MS analysis confirmed that genetic transformation did not lead to changes in the content of the main secondary metabolites of apple roots. Dihydrochalcones phloridzin and phloretin, chlorogenic and caffeic acids were detected as only putative allelochemicals exuded into the growth medium in which hairy roots were grown. Apple hairy root exudates significantly affected development and growth of Arabidopsis thaliana seedlings. Pure phloridzin at 500 μM and phloretin in all tested concentrations (125-500 μM) had similar effects as apple hairy root exudates inducing significant alterations in morphology of seedlings, expecially roots. High similarities in the expression patterns of genes involved in polar auxin transport and auxin and gibberellin biosynthetic pathways in roots of arabidopsis seedlings treated with either apple hairy root exudates or 500 μM phloretin were observed. This indicates that phloretin in hairy root exudates could be at least partly responsible for the apple root allelopathic properties, that were most likely based on auxin and gibberellin cross-talk and their altered homeostasis in arabidopsis roots.

Apple dihydrochalcones phloridzin and phloretin as novel allelochemicals

Sekundarni metaboliti jabuke (Malus × domestica Borkh.) florizin i floretin su dihidrohalkoni visoko specifični za vrste roda Malus. Autoalelopatski potencijal ovih fenolnih jedinjenja se ogleda u suzbijanju rasta klijanca i sadnica zasađenih na mestu prvobitnih voćnjaka jabuke. U osnovi njihove fitotoksičnosti su ultrastrukturne i molekularne promene koje se ogledaju u povećanju sadržaja malondialdehida, prolina i aktivnosti antioksidativnih enzima: superoksid-dismutaze, peroksidaza i katalaza, kao i nivoa ekspresije proteina uključenih u odbrambene mehanizme ćelije. Najnovija istraživanja ukazuju na fitotoksičnost ovih dihidrohalkona i prema drugim biljnim vrstama, kao npr. korovskoj model biljci Arabidopsis thaliana. Tretman floretinom izaziva značajnu inhibiciju rastenja i razvića klijanaca A. thaliana, poremećaj gravitropskog odgovora i pojavu morfoloških i ultrastrukturnih malformacija u čijoj osnovi leži promena ekspresije CDKA1;1, CDKB2;1, CYCA3;1 i CYCB2;4 gena ćelijskog ciklusa, kao i gena uključenih u održanje homeostaze auksina (PINs, AUX1, LAX3, ABCBs, TAA1 i YUCs) i biosinteze giberelina (GA20ox2 i GA3ox1). Ovo otvara mogućnost za dalja istraživanja njihovog alelopatskog delovanja ali i potencijalnu primenu kao ekološki bezbednih bioherbicida.Phloridzin and phloretin are dihydrochalcones highly specific to domestic apple (Malus × domestica Borkh.) and other species from genus Malus. Auto-allelopathic potential of these phenolics was recognized in ‘Apple Replant Disease’ (ARD) characterized by stunted growth, reduced root system and low yields in apple trees replanted on lands that previously supported apple orchards. Physiological basis of phloridzin and phloretin autotoxicity are ultrastructural and molecular alterations that include increased catalase, peroxidases and superoxide dismutase activities, raised malondialdehyde and proline contents, and high expression of genes involved in cell defence system. The latest research on model plant Arabidopsis thaliana indicates phytotoxicity of these dihydrochalcones towards other plant species. Phloretin inhibits A. thaliana seedlings growth and development inducing agravitropic phenotype and morphological and ultrastructural malformations in treated seedlings. Altered expression of CDKA1;1, CDKB2;1, CYCA3;1 and CYCB2;4 cell cycle genes and genes involved in auxin homeostasis (PINs, AUX1, LAX3, ABCBs, TAA1 and YUCs) and gibberellin biosynthesis (GA20ox2 and GA3ox1) are in the physiological bases of phloretin phytotoxicity. This mechanism makes phloretin a prospective candidate for an eco-friendly bioherbicide and paves the way for further research of phloretin role in ARD

The effects of β-lactam antibiotics and hygromycin B on de novo shoot organogenesis in apple cv. Golden Delicious

Since the genetic transformation of the apple is strongly genotype-dependent and generally inefficient, the evaluation of factors affecting shoot regeneration are crucial for the establishment of a successful transformation process. In this report, we evaluated the effects of the β-lactam antibiotics meropenem and timentin on in vitro regeneration via de novo shoot organogenesis from leaf explants of apple cv. Golden Delicious, as well as on the growth of the Agrobacterium tumefaciens strain EHA 105, and compared them with the commonly used β-lactam cefotaxime. Also, we report for the first time the effect of hygromycin B as a selective agent in the domesticated apple, as regards shoot regeneration and shoot multiplication efficiency. We observed that cefotaxime and timentin at concentrations higher than 100 mg L-1 were sufficient to prevent Agrobacterium growth during a two-week period, while meropenem exhibited an inhibitory effect on bacterial growth at all tested concentrations (25-150 mg L-1). Cefotaxime at a concentration of 300 mg L-1 increased the number of regenerated shoots per explant (9.39) in comparison with the control (7.67). In contrast to cefotaxime, meropenem and timentin caused a decrease in shoot regeneration efficiency, but larger and more developed shoots were obtained on meropenem (25-125 mg L-1) after the same period of cultivation. Hygromycin B at a concentration of 5 mg L-1 or higher completely inhibited shoot regeneration and induced explant tissue necrosis. Therefore, the selection procedure with a final concentration of 4 mg L-1 throughout organogenesis and 10 mg L-1 for further shoot growth and multiplication is recommended for an efficient transformation process in apple cv. Golden Delicious

A Metabonomic View on Wilms Tumor by High-Resolution Magic-Angle Spinning Nuclear Magnetic Resonance Spectroscopy

Pediatric cancer NMR-metabonomics might be a powerful tool to discover modified biochemical pathways in tumor development, improve cancer diagnosis, and, consequently, treatment. Wilms tumor (WT) is the most common kidney tumor in young children whose genetic and epigenetic abnormalities lead to cell metabolism alterations, but, so far, investigation of metabolic pathways in WT is scarce. We aimed to explore the high-resolution magic-angle spinning nuclear magnetic resonance (HR-MAS NMR) metabonomics of WT and normal kidney (NK) samples. For this study, 14 WT and 7 NK tissue samples were obtained from the same patients and analyzed. One-dimensional and two-dimensional HR-MAS NMR spectra were processed, and the one-dimensional NMR data were analyzed using chemometrics. Chemometrics enabled us to elucidate the most significant differences between the tumor and normal tissues and to discover intrinsic metabolite alterations in WT. The metabolic differences in WT tissues were revealed by a validated PLS-DA applied on HR MAS T2 -edited 1H-NMR and were assigned to 16 metabolites, such as lipids, glucose, and branched chain amino acids (BCAAs), among others. The WT compared to NK samples showed 13 metabolites with increased concentrations and 3 metabolites with decreased concentrations. The relative BCAA concentrations were decreased in the WT while lipids, lactate, and glutamine/glutamate showed increased levels. Sixteen tissue metabolites distinguish the analyzed WT samples and point to altered glycolysis, glutaminolysis, TCA cycle, and lipid and BCAA metabolism in WT. Significant variation in the concentrations of metabolites, such as glutamine/glutamate, lipids, lactate, and BCAAs, was observed in WT and opened up a perspective for their further study and clinical validation

Physiological and cell ultrastructure disturbances in wheat seedlings generated by Chenopodium murale hairy root exudate.

Chenopodium murale L. is an invasive weed species significantly interfering with wheat crop. However, the complete nature of its allelopathic influence on crops is not yet fully understood. In the present study, the focus is made on establishing the relation between plant morphophysiological changes and oxidative stress, induced by allelopathic extract. Phytotoxic medium of C. murale hairy root clone R5 reduced the germination rate (24% less than control value) of wheat cv. Nataša seeds, as well as seedling growth, diminishing shoot and root length significantly, decreased total chlorophyll content, and induced abnormal root gravitropism. The R5 treatment caused cellular structural abnormalities, reflecting on the root and leaf cell shape and organization. These abnormalities mostly included the increased number of mitochondria and reorganization of the vacuolar compartment, changes in nucleus shape, and chloroplast organization and distribution. The most significant structural changes were observed in cell wall in the form of amoeboid protrusions and folds leading to its irregular shape. These structural alterations were accompanied by an oxidative stress in tissues of treated wheat seedlings, reflected as increased level of H2O2 and other ROS molecules, an increase of radical scavenging capacity and total phenolic content. Accordingly, the retardation of wheat seedling growth by C. murale allelochemicals may represent a consequence of complex activity involving both cell structure alteration and physiological processes.This is a post-peer-review, pre-copyedit version of an article published in Protoplasma. The final authenticated version is available online at: [http://dx.doi.org/10.1007/s00709-018-1250-0

Allelopathic potential of apple hairy root secondary metabolites - the effect of phloretin and phloridzin in vitro culture

Ograniĉenja u izuĉavanju potencijala i mehanizama alelopatskog delovanja glavnih sekundarnih metabolita korena jabuke, floretina i florizina, usled kompleksnosti interakcija u rizosferi, mogu biti prevaziĊena upotrebom kulture transformisanih korenova in vitro. Odabranim naĉinom inokulacije regenerisanih izdanaka jabuke sa Agrobacterium rhizogenes 15834, efikasno su indukovani transformisani korenovi kod sorti Melrouz, Zlatni Delińes, Ĉadel i Gloster. Smenom hranljivih podloga sa i bez auksina, po prvi put je uspostavljena permanentna in vitro kultura izolovanih transformisanih korenova jabuke. UHPLC/(+/-)HESI–MS/MS analizom je utvrĊeno da genetiĉka transformacija nije dovela do promena u sadrņaju glavnih sekundarnih metabolita korena jabuke. U eksudatu transformisanih korenova, sadrņanom u teĉnoj podlozi u kojoj su korenovi gajeni, kvantifikovani su iskljuĉivo florizin, floretin, hlorogena i kafeinska kiselina. Tretman ovim eksudatom je prouzrokovao znaĉajnu inhibiciju rastenja i razvića izdanaka i korenova klijanaca Arabidopsis thaliana. Sliĉan inhibitorni efekat ispoljili su ĉist florizin u koncentraciji od 500 μM, kao i floretin u svim testiranim koncentracijama (125-500 μM), izazivajući krupne promene u morfologiji tretiranih klijanaca, posebno korena. Velike sliĉnosti su zapaņene u obrascu promena ekspresije gena ukljuĉenih u polarni transport auksina, kao i biosintezu auksina i giberelina u korenovima klijanaca arabidopsisa tretiranih eksudatom transformisanih korenova jabuke, sa onim detektovanim kod klijanaca tretiranih 500 μM floretinom. To ukazuje na mogućnost da je floretin u eksudatu korena jabuke metabolit koji je odgovoran za alelopatske efekte, a koji bi se mogli zasnivati na promenama nivoa auksina i giberelina, kao i na interakciji ove dve grupe hormona

The effects of β-lactam antibiotics and hygromycin B on de novo shoot organogenesis in apple cv. Golden Delicious

Since the genetic transformation of the apple is strongly genotype-dependent and generally inefficient, the evaluation of factors affecting shoot regeneration are crucial for the establishment of a successful transformation process. In this report, we evaluated the effects of the β-lactam antibiotics meropenem and timentin on in vitro regeneration via de novo shoot organogenesis from leaf explants of apple cv. Golden Delicious, as well as on the growth of the Agrobacterium tumefaciens strain EHA 105, and compared them with the commonly used β-lactam cefotaxime. Also, we report for the first time the effect of hygromycin B as a selective agent in the domesticated apple, as regards shoot regeneration and shoot multiplication efficiency. We observed that cefotaxime and timentin at concentrations higher than 100 mg L-1 were sufficient to prevent Agrobacterium growth during a two-week period, while meropenem exhibited an inhibitory effect on bacterial growth at all tested concentrations (25-150 mg L-1). Cefotaxime at a concentration of 300 mg L-1 increased the number of regenerated shoots per explant (9.39) in comparison with the control (7.67). In contrast to cefotaxime, meropenem and timentin caused a decrease in shoot regeneration efficiency, but larger and more developed shoots were obtained on meropenem (25-125 mg L-1) after the same period of cultivation. Hygromycin B at a concentration of 5 mg L-1 or higher completely inhibited shoot regeneration and induced explant tissue necrosis. Therefore, the selection procedure with a final concentration of 4 mg L-1 throughout organogenesis and 10 mg L-1 for further shoot growth and multiplication is recommended for an efficient transformation process in apple cv. Golden Delicious. [Project of the Serbian Ministry of Education, Science and Technological Development, Grant no. ON173015

Diurnal rhythmicity of endogenous phytohormones and phototropic bending capacity in potato (Solanum tuberosum L.) shoot cultures

Taking advantage of advanced high performance liquid chromatography–electrospray tandem–mass spectrometry (HPLC–ESI–MS/MS), we screened daily changes in concentrations of endogenous phytohormones of in vitro grown potato (Solanum tuberosum L. cv. Désirée) shoot cultures and checked for possible connections between the rhythmicity of endogenous phytohormones and phototropic bending capacity of the same cultures. Studies done under diurnal 16 h light and 8 h darkness (diurnal) and continuous light (CL) conditions showed prominent daily rhythmicity of endogenous phytohormone levels in both light regimes. Phototropic bending in potato is known to be rhythmic only in the diurnal, whereas in CL conditions the bending response is present but without any daily rhythmicity. For all of the studied phytohormone groups significant differences between the diurnal and CL conditions were found. Changes in the concentration of indole auxins, indole-3-acetic acid (IAA) and its catabolite 2-oxindole-3-acetic acid (OxIAA), were the most prominent. Their levels clearly alternated with level of IAA being high in diurnal and OxIAA in CL conditions. Significant concentration changes were also observed for other phytohormones such as cytokinin ribosides, salicylic acid, abscisic acid and phaseic acid. Observed changes in daily phytohormone levels indicate strong and complex involvement of diverse phytohormone groups in realization of the phototropic bending response of potato shoots

Overcoming Low Germination and Low Quality of Flax Seeds (<i>Linum usitatissimum</i> L.) in Unfavorable Storage Using Static Magnetic Fields

Flax seeds stored in unfavorable conditions were exposed to static magnetic fields (SMFs) of 50–350 mT for 20–120 min to overcome low germination and quality. Seed germination increased slightly with increasing strength of SMF and duration of treatment. Seed germination from 89% to 100% was achieved in SMF treatments of 150 mT (120 min), 200 mT (80–120 mT), 250 mT (60–120 min), 300 mT (40–120 min), and 350 mT (40–120 min). In these treatments, germination was 2.78- to 3.12-fold higher than in the control after 10 days. Treatments with 350 mT for 100 and 120 min showed the best results in germination (100%), as well as a 26.81-fold increase in vigor I, 28.69-fold increase in vigor II, 1-fold increase in chlorophyll a, 0.84-fold in chlorophyll b, 0.46-fold increase in carotenoid content, and 2.63-fold increase in catalase activity compared to the control after 10 days. Also, SMF treatment of 350 mT (20–120 min) reduced cell leakage and electrical conductivity by 1-fold compared to the control. SMF is a healthy, biologically safe, and environmentally friendly treatment and can be a tool for overcoming problems of low germination and quality of seeds stored under unfavorable conditions